An Autonomously Replicating Sequence (ARS) is a sequence that serves as an origin of DNA replication on eukaryotic chromosomes. An ARS is required for DNA replication during cell division, and is a necessary part of a functional synthetic chromosome (Gilbert (2001) Science 294:96-100). Additionally, some ARS sequences have been shown to function as matrix attachment regions (MARs), which can improve heterologous genes' amount of expression, consistency of expression over time, and degree of repression by heterochromatin formation (Kim et al. 2007).
While DNA sequences that constitute the origins of DNA replication are readily identifiable in bacteria, origins of replication in eukaryotes have been more difficult to identify (Gilbert 2001). ARS primary sequences are not significantly conserved, either within or across eukaryotic species, although the location of some ARS elements is conserved across species (Gilbert 2001). For example, Saccharomyces cerevisiae (budding yeast) (Smith & Boeke (1997) Genes Dev. 11:241-54) and Pisum sativum (garden pea) (Van't Hof (1996) “DNA Replication in Eukaryotic Cells,” CSHL Press) both have an ARS between the 18S and 5S ribosomal RNA genes in their respective ribosomal repeat regions.
The current methodology for acquiring non-Saccharomyces cerevisiae ARS elements for use in artificial chromosomes is through random acquisition with large (˜100 kbp) pieces of genomic DNA (Basu et al. (2005) BMC Biotechnol. 5:21). This process is both inefficient and liable to import sequences that are deleterious to optimal industrial performance, but which happen to be located proximal on a chromosome to an ARS. Once an ARS is located, it can be placed into artificial chromosomes or plasmids to permit those constructs to replicate in a host of interest (Henning et al. (1999) Proc. Nat'l. Acad. Sci. USA 96:592-97). At present, there are no known Nannochloropsis sp.—or heterokont—ARS elements available.